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- PDB-6l4a: H3-H3-H3 tri-nucleosome with the 22 base-pair linker DNA -

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Basic information

Entry
Database: PDB / ID: 6l4a
TitleH3-H3-H3 tri-nucleosome with the 22 base-pair linker DNA
Components
  • (DNA (485-MER)) x 2
  • Histone H2A type 1-B/E
  • Histone H2B type 1-J
  • Histone H3.1Histone H3
  • Histone H4
KeywordsNUCLEAR PROTEIN / Chromatin / Nucleosome / Centromere
Function / homology
Function and homology information


negative regulation of megakaryocyte differentiation / CENP-A containing nucleosome assembly / DNA replication-independent nucleosome assembly / telomere capping / negative regulation of tumor necrosis factor-mediated signaling pathway / interleukin-7-mediated signaling pathway / innate immune response in mucosa / DNA-templated transcription, initiation / telomere organization / DNA replication-dependent nucleosome assembly ...negative regulation of megakaryocyte differentiation / CENP-A containing nucleosome assembly / DNA replication-independent nucleosome assembly / telomere capping / negative regulation of tumor necrosis factor-mediated signaling pathway / interleukin-7-mediated signaling pathway / innate immune response in mucosa / DNA-templated transcription, initiation / telomere organization / DNA replication-dependent nucleosome assembly / nuclear nucleosome / chromatin silencing at rDNA / negative regulation of gene expression, epigenetic / regulation of gene silencing by miRNA / nuclear chromosome / regulation of gene silencing / regulation of megakaryocyte differentiation / protein heterotetramerization / nucleosome assembly / nucleosome / lipopolysaccharide binding / double-strand break repair via nonhomologous end joining / chromatin organization / nuclear chromosome, telomeric region / antibacterial humoral response / killing of cells of other organism / antimicrobial humoral immune response mediated by antimicrobial peptide / protein ubiquitination / blood coagulation / cadherin binding / defense response to Gram-negative bacterium / defense response to Gram-positive bacterium / negative regulation of cell population proliferation / nuclear chromatin / protein domain specific binding / cellular protein metabolic process / protein heterodimerization activity / protein-containing complex / RNA binding / DNA binding / extracellular space / extracellular exosome / membrane / nucleoplasm / extracellular region / nucleus / cytosol
C-terminus of histone H2A / Centromere kinetochore component CENP-T histone fold / Core histone H2A/H2B/H3/H4 / CENP-T/Histone H4, histone fold / Histone H2A conserved site / Histone H2A, C-terminal domain / Histone H4, conserved site / Histone-fold / Histone H2A/H2B/H3 / TATA box binding protein associated factor (TAF) ...C-terminus of histone H2A / Centromere kinetochore component CENP-T histone fold / Core histone H2A/H2B/H3/H4 / CENP-T/Histone H4, histone fold / Histone H2A conserved site / Histone H2A, C-terminal domain / Histone H4, conserved site / Histone-fold / Histone H2A/H2B/H3 / TATA box binding protein associated factor (TAF) / Histone H2A / Histone H4 / Histone H2B / Histone H3/CENP-A
Histone H2A type 1-B/E / Histone H2B type 1-J / Histone H4 / Histone H3.1
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 12.3 Å
AuthorsTakizawa, Y. / Ho, C.-H. / Tachiwana, H. / Matsunami, H. / Ohi, M. / Wolf, M. / Kurumizaka, H.
Funding support Japan, 6items
OrganizationGrant numberCountry
Japan Society for the Promotion of ScienceJP18H05534 Japan
Japan Society for the Promotion of ScienceJP17H01408 Japan
Japan Society for the Promotion of ScienceJP17H05013 Japan
Japan Society for the Promotion of Science17H06167 Japan
Japan Society for the Promotion of Science15H05972 Japan
Japan Society for the Promotion of ScienceJP19K06522 Japan
CitationJournal: Structure / Year: 2019
Title: Cryo-EM Structures of Centromeric Tri-nucleosomes Containing a Central CENP-A Nucleosome.
Authors: Yoshimasa Takizawa / Cheng-Han Ho / Hiroaki Tachiwana / Hideyuki Matsunami / Wataru Kobayashi / Midori Suzuki / Yasuhiro Arimura / Tetsuya Hori / Tatsuo Fukagawa / Melanie D Ohi / Matthias Wolf / Hitoshi Kurumizaka /
Abstract: The histone H3 variant CENP-A is a crucial epigenetic marker for centromere specification. CENP-A forms a characteristic nucleosome and dictates the higher-order configuration of centromeric ...The histone H3 variant CENP-A is a crucial epigenetic marker for centromere specification. CENP-A forms a characteristic nucleosome and dictates the higher-order configuration of centromeric chromatin. However, little is known about how the CENP-A nucleosome affects the architecture of centromeric chromatin. In this study, we reconstituted tri-nucleosomes mimicking a centromeric nucleosome arrangement containing the CENP-A nucleosome, and determined their 3D structures by cryoelectron microscopy. The H3-CENP-A-H3 tri-nucleosomes adopt an untwisted architecture, with an outward-facing linker DNA path between nucleosomes. This is distinct from the H3-H3-H3 tri-nucleosome architecture, with an inward-facing DNA path. Intriguingly, the untwisted architecture may allow the CENP-A nucleosome to be exposed to the solvent in the condensed chromatin model. These results provide a structural basis for understanding the 3D configuration of CENP-A-containing chromatin, and may explain how centromeric proteins can specifically target the CENP-A nucleosomes buried in robust amounts of H3 nucleosomes in centromeres.
Validation Report
SummaryFull reportAbout validation report
History
DepositionOct 16, 2019Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 4, 2019Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: Histone H3.1
B: Histone H4
C: Histone H2A type 1-B/E
D: Histone H2B type 1-J
E: Histone H3.1
F: Histone H4
G: Histone H2A type 1-B/E
H: Histone H2B type 1-J
I: DNA (485-MER)
J: DNA (485-MER)
K: Histone H3.1
L: Histone H4
M: Histone H2A type 1-B/E
N: Histone H2B type 1-J
O: Histone H3.1
P: Histone H4
Q: Histone H2A type 1-B/E
R: Histone H2B type 1-J
S: Histone H3.1
T: Histone H4
U: Histone H2A type 1-B/E
V: Histone H2B type 1-J
W: Histone H3.1
X: Histone H4
Y: Histone H2A type 1-B/E
Z: Histone H2B type 1-J


Theoretical massNumber of molelcules
Total (without water)635,98526
Polymers635,98526
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein/peptide , 4 types, 24 molecules AEKOSWBFLPTXCGMQUYDHNRVZ

#1: Protein/peptide
Histone H3.1 / Histone H3 / Histone H3/a / Histone H3/b / Histone H3/c / Histone H3/d / Histone H3/f / Histone H3/h / Histone H3/i / Histone H3/j / Histone H3/k / Histone H3/l


Mass: 15719.445 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Gene: HIST1H3A, H3FA, HIST1H3B, H3FL, HIST1H3C, H3FC, HIST1H3D, H3FB, HIST1H3E, H3FD, HIST1H3F, H3FI, HIST1H3G, H3FH, HIST1H3H, H3FK, HIST1H3I, H3FF, HIST1H3J, H3FJ
Production host: Escherichia coli (E. coli) / References: UniProt: P68431
#2: Protein/peptide
Histone H4 /


Mass: 11676.703 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Gene: HIST1H4A, H4/A, H4FA, HIST1H4B, H4/I, H4FI, HIST1H4C, H4/G, H4FG, HIST1H4D, H4/B, H4FB, HIST1H4E, H4/J, H4FJ, HIST1H4F, H4/C, H4FC, HIST1H4H, H4/H, H4FH, HIST1H4I, H4/M, H4FM, HIST1H4J, H4/E, H4FE, HIST1H4K, H4/D, H4FD, HIST1H4L, H4/K, H4FK, HIST2H4A, H4/N, H4F2, H4FN, HIST2H4, HIST2H4B, H4/O, H4FO, HIST4H4
Production host: Escherichia coli (E. coli) / References: UniProt: P62805
#3: Protein/peptide
Histone H2A type 1-B/E / Histone H2A.2 / Histone H2A/a / Histone H2A/m


Mass: 14447.825 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HIST1H2AB, H2AFM, HIST1H2AE, H2AFA / Production host: Escherichia coli (E. coli) / References: UniProt: P04908
#4: Protein/peptide
Histone H2B type 1-J / Histone H2B.1 / Histone H2B.r / H2B/r


Mass: 14217.516 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HIST1H2BJ, H2BFR / Production host: Escherichia coli (E. coli) / References: UniProt: P06899

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DNA chain , 2 types, 2 molecules IJ

#5: DNA chain DNA (485-MER)


Mass: 149214.703 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#6: DNA chain DNA (485-MER)


Mass: 150401.328 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: H3-H3-H3 tri-nucleosome with the 22 base-pair linker DNA
Type: COMPLEX / Entity ID: 1,2,3,4,5,6 / Source: RECOMBINANT
Molecular weightValue: 0.6 kDa/nm / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.8
SpecimenDetails: H3-H3-H3 tri-nucleosome with the 22 base-pair linker DNA
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 80 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: (1.14_3260: phenix.real_space_refine) / Classification: refinement
EM software
IDNameVersionCategory
4CTFFIND4CTF correction
10RELION2.1initial Euler assignment
11RELION2.1final Euler assignment
12RELION2.1classification
13RELION2.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 12.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 7312 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT

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